Mass spectrometry quantitation of p450 protein isoforms in hepatocytes

ABSTRACT

A method for screening a drug for cytochrome P450 (CYP) induction is provided and can include incubating the drug with a microsome-containing biological sample and then quantitating at least one cytochrome P450 isoform. The isoforms can be selected from 2B6, 3A4, 1A2, and 3A5 isoforms. In some embodiments, the method uses liquid chromatography tandem mass spectrometry (LC-MSMS). A quantitated value can be compared to a threshold value and the drug can be determined to exhibit an acceptable CYP induction potential when the quantitated value does not exceed the threshold value. Isolated peptides are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.12/904,520, filed Oct. 13, 2010, which claims the benefit of priorityfrom U.S. Provisional Patent Application Nos. 61/252,648, filed Oct. 17,2009, and 61/252,430, filed Oct. 16, 2009, all of which are incorporatedherein in their entireties by reference.

FIELD

The present teachings relate to cytochrome P450 enzymes (CYPs) anddetection of enzymes using mass spectrometry.

BACKGROUND

Cytochrome P450 enzymes (CYPs) are major drug metabolizing enzymes andexperimental pharmaceutical compounds are generally evaluated for theirCYP induction potential early in the development process. Measurement ofa CYP induction profile in response to a chemical can be used as afundamental aspect of drug safety evaluation, but expression of theseproteins is regulated by transcriptional, post transcriptional andtranslational mechanisms. As a result, mRNA-based assays are notreliable predictors of CYP induction. Further, CYPs exhibit extensiveamino acid sequence homology, particularly within subfamilies, so thatP450 protein expression methods are also poorly discriminatory.

SUMMARY

According to various embodiments of the present teachings, a method ofquantitating the 1A2, 2B6, 3A4, and 3A5 isoforms of cytochrome P450enzymes (CYPs) is provided without the need for any chemical labeling.Isoform-specific tryptic peptides can be observed in liquidchromatography-Tandem Mass Spectrometry (LC-MSMS) analysis of samplesderived from hepatocytes, for example, in microsomes, along with theiroptimal Q1 and Q3 transitions. Those observed peptides and transitions,can be used to enable a reliable CYP quantitation of the isoforms 1A2,2B6, 3A4, and 3A5.

According to various embodiments, a set of peptides and optimal MRMtransistors are provided as “house keeping” microsomol proteins whoseconcentrations are unaffected by drug incubation. The set can be used asnormalization proteins for quantitative analysis. In some embodiments,quantitation can be performed by spiking into the sample heavy forms ofthe isoform-specific peptides, for example, forms that have beenenriched with C13 and/or N15. FIG. 1 shows a typical MRM analysis forCYP 1A2, CYP2B6, and CYP3A3/3A4 from human microsomes, wherein some ofthe isoform-specific peptides have been labeled in FIG. 1. The optimizedtransitions for each of a set of observed, most sensitiveisoform-specific peptides, according to various embodiments of thepresent teachings, is shown in Tables 1-4 below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will be described with reference to theaccompanying drawings. The drawings are intended to illustrate, notlimit, the present teachings.

FIG. 1 shows an MRM analysis of 20 μg of a microsomal preparation forCYP 1A2, CYP 2B6, and CYP 3A4 (extracted ion chromatograms (XIC) of +MRM(412 pairs)).

FIG. 2 shows an MRM analysis of 1 μl of a microsomal preparation for CYP2B6 (extracted ion chromatogram of +MRM (15 pairs)).

FIG. 3 shows an MRM analysis of 2 μg of a microsomal preparation for CYP1A2 (extracted ion chromatogram of +MRM (57 pairs)).

FIG. 4 shows an MRM analysis of 1 μl of a microsomal preparation for CYP3A4 (extracted ion chromatogram of +MRM (34 pairs)).

FIG. 5A shows an MRM analysis for 30 μl of a microsomal preparation for3A5 (extracted ion chromatogram of +MRM (48 pairs)).

FIG. 5B shows an MRM analysis for 30 μl of a microsomal preparation for3A5 (extracted ion chromatogram of +MRM (48 pairs)).

FIG. 5C shows an MRM analysis for 30 μl of a microsomal preparation forA5 (extracted ion chromatogram of +MRM (48 pairs)).

FIG. 6 shows a graph showing protein expression changes observed in ahepatocyte sample preparation treated with inducers for CYP 2B6, usingRNA assays, enzyme activity assays and the protein quantitation methodof the present teachings.

FIG. 7 shows a graph showing protein expression changes observed in ahepatocyte sample preparation treated with inducers far CYP 1A2, usingRNA assays, enzyme activity assays and the protein quantitation methodof the present teachings.

FIG. 8 shows a graph showing protein expression changes observed in ahepatocyte sample preparation treated with inducers far CYP 3A4, usingRNA assays, enzyme activity essays and the protein quantitation methodof the present teachings.

DETAILED DESCRIPTION

According to various embodiments, a method for screening a drug forcytochrome P450 (CYP) induction is provided that comprises incubatingthe drug with a microsome-containing biological sample and quantitatingat least one CYP isoform. In some embodiments, the isoforms con compriseone or more isoform selected front 2B6, 3A4, 1A2, and 3A5 isoforms. Themethod can comprise using a liquid chromatography tandem massspectrometry (LC-MSMS) technique to quantitate the amount of eachisoform. The quantitated value of each can be compared to a thresholdvalue, and the drug can be identified as having an acceptable CYPinduction potential when the quantitated value does not exceed thethreshold value. The threshold value can be selected or predeterminedbased on a desired CYP induction potential or based on the CYP inductionpotential of one or more different drugs, similar or non-similar to thedrug being screened. In some embodiments, the microsome-containingbiological sample can be derived from a mammal, from a primate, or froma human.

According to various embodiments, the drug can be incubated with asample containing human hepatocytes. In some embodiments, afterincubation with the drag, the sample containing human hepatocytes can beused to obtain at least one microsome fraction by, for example, 16 Gcentrifugation. The microsome fraction can be analyzed for CYP inductionby the drug, by detecting isolated peptides specific to CYP(isoform-specific peptides). According to some embodiments, afterincubation with the drug, the sample containing human hepatocytes can beused to obtain at least one S9 fraction by, for example, 9 Gcentrifugation. The S9 fraction can be analyzed to detect CYP inductionby the drug, for example, by detecting isolated peptides specific to CYP(isoform-specific peptides). According to some embodiments, themicrosome fraction or the S9 faction can be analyzed using a liquidchromatography tandem mass spectrometry (LC-MSMS) technique in order toquantitate at least one CYP isoform. The quantitated value of each canbe compared to a threshold value, and the drug can be identified ashaving an acceptable CYP induction potential when the quantitated valuedoes not exceed the threshold value.

According to various embodiments, a method is provided for directlyanalyzing CYP from hepatocytes. In some embodiments, antibody peptidescan be used to pull the isoform-specific peptides directly out ofhepatocyctes. According to some embodiments, using antibody peptides topull the isoform-specific peptides directly out of hepatocytes wouldhave the advantage of not needing to prepare S9 or microsome fractions,and would require less hepatocyte cells for drug incubation.

In some embodiments, the method comprises comparing detected inductionto a control. For example, became little or no drug induction of CYPs isdesirable, a threshold can be set such that the drug mug show less than(<) 40% induction compared to the positive control, to be consideredacceptable. In some embodiments, the drug must show less than (<) 30%induction compared to the positive control, to be considered acceptable.In other embodiments, the drug mast show less than (<) 20% inductioncompared to the positive control, to be considered acceptable.

According to various embodiments, a method for determining an amount ofat least one isoform of cytochrome P450 (CYP) in a sample, is provided.The method can comprise the use of a mass spectrometry technique,wherein the at least one isoform of cytochrome P450 comprises at leastone of CYP 2B6, CYP 3A4, CYP 1A2, and CYP 3A5. The mass spectrometrytechnique can comprise a tandem mass spectrometry (MS/MS) techniqueand/or a liquid chromatography tandem mass spectrometry (LC-MS/MS)technique. In some embodiments, the technique comprises an LC-MS/MStechnique and the use of a triple quadrupole instrument and MultipleReaction Monitoring (MRM).

FIG. 1 shows a typical MRM analysis for CYP 1A2, CYP 2B6, and CYP 3A4,well as the “housekeeping” Microsomal proteins (Microsomal GST,Corticosteroid 11 beta, and Microsomal Tryglyceride), from a microsomalsample preparation prepared as described below in the Examples. Thequantity of CYP 1A2, CYP 2B6, and CYP 3A4 can be determined by, forexample, Isotope Dilution Mass Spectrometry, wherein the samplepreparation is spiked with heavy forms of the isoform-specific peptides.The quantity of CYP 1A2, CYP 2B6, and CYP 3A4 can also be determinedusing other conventional methods known in the art. In some embodiments,the method uses LC-MSMS with multiple reaction monitoring (MRM)quantitation of the isoform-specific peptides and isotope-coded affinitytags (ICAT) to generate a CYP induction profile. The method can use, forexample, approaches similar to the approaches presented by Pennington etal. to quantitate isoform-specific cysteine-containing peptides labeledwith ICAT as described in Proteomics, 6(6), pages 1934-1947 (March2006), which is incorporated herein in its entirety by reference.

FIG. 2 shows a typical MRM analysis for CYP 2B6 from the microsomalpreparation prepared a described herein. Isolated peptides comprisingthe amino acid sequence of SEQ ID NOS: 1, 2, 3, or 4 identified in Table1 below are specific to CYP 2B6.

FIG. 3 shows a typical MRM analysis for CYP 1A2 from the microsomalpreparation prepared as described herein. The isolated peptidescomprising the amino acid sequence of SEQ ID NOS: 8, 9, 10, 11, 12, 13,or 14 identified in Table 3 below are specific CYP 1A2.

FIG. 4 shows a typical MRM analysis for CYP 3A4 from the microsomalpreparation prepared as described herein. The isolated peptidescomprising the amino acid sequence of SEQ ID NOs: 5, 6, or 7 identifiedin Table 2 below are specific to CYP 3A4. It should be understood thatpeptides comprising the amino acid sequence of SEQ ID NOS: 5, 6, or 7can also be used to identify and/or quantify CYP 3A3.

The isolated peptides comprising the amino acid sequence of SEQ ID NOs:15, 15, or 17 identified in Table 4 below are specific to CYP 3A5.

FIGS. 5A-5C show three different panes of a typical MRM analysis for CYP3A5 from the microsomal preparation prepared as described herein. Eachpane relates to specific transitions used for the particular peptide.The isolated peptides comprising the amino acid sequence or SEQ ID NOS:15, 16 or 17 are specific to CYP 3A5.

According to various embodiments, the method can comprise determining anamount or CYP 2B6 in the sample by detecting an isolated peptidespecific to cytochrome P450 (CYP) isoform CYP 2B6, for example, one ormore of the isoforms comprising the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3 identified herein. The amount can bedetermined using a triple quadrupole instrument and Multiple ReactionMonitoring (MRM). In some embodiments, the isolated peptide can comprisethe amino acid sequence of SEQ ID NO: 1 identified herein, and themethod can comprise monitoring precursor-product ion pair transitionshaving an m/z value of about 548/911, 548/681, or 548/566, wherein theterm “about” as used herein means within a range of +/− one (1) atomicmass unit. In some embodiments, the isolated peptide can comprise theamino acid sequence of SEQ ID NO: 2 identified herein, and the methodcan comprise monitoring precursor-product ion pair transitions having anm/z value of about 494/777, 494/437, or 494/874. In some embodiments,the isolated peptide can comprise the amino acid sequence of SEQ ID NO:3 identified herein, and the method can comprise monitoringprecursor-product ion pair transitions having an m/z value of about421/508, 421/607, or 421/694. In some embodiments, the isolated peptidecan comprise the amino acid sequence of SEQ ID NO: 4, and the method cancomprise monitoring precursor-product ion pair transitions having an m/zvalue of about 479/499, 479/614, or 479/727.

According to various embodiments, the method can comprise determining anamount of CYP 3A4 in the sample by detecting an isolated peptidespecific to cytochrome P450 (CYP) isoform CYP 3A4, comprising the aminoacid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 identifiedherein. The method can use, for example, a triple quadrupole instrumentand Multiple Reaction Monitoring (MRM). In some embodiments, theisolated peptide can comprise the amine acid sequence of SEQ ID NO: 5identified herein, and the method can comprise monitoringprecursor-product ion pair transitions having an m/z value of about440/549, 440/650, or 440/532. In some embodiments, the isolated peptidecan comprise the amino acid sequence of SEQ ID NO: 6 identified herein,and the method can comprise monitoring precursor-product ion pairtransitions having an m/z value of about 704/794, 704/929, 564/639,564/745, or 564/790. In some embodiments, the isolated peptide cancomprise the amino acid sequence of SEQ ID NO: 7 identified herein, andthe method can comprise monitoring precursor-product ion per transitionshaving an m/z value or about 798/819, 798/932, or 798/1004.

According to various embodiments, the method can comprise determining enamount of CYP 1A2 in the sample by detecting an isolated peptidespecific to cytochrome P450 (CYP) isobar CYP 1A2, comprising the aminoacid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, identified herein.The method can use, for example, a triple quadrupole instrument andMultiple Reaction Monitoring (MRM).

In some embodiments, the isolated peptide can comprise the amino acidsequence of SEQ ID NO: 8 identified herein, and the method can comprisemonitoring precursor-product ion pair transitions having an m/z value ofabout 432/636, 432/535, or 432/478. In some embodiments, the isolatedpeptide can comprise the amino acid sequence of SEQ ID NO: 9 identifiedherein, and the method can comprise monitoring precursor-product ionpair transitions having an m/z value of about 482/800, 482/628, or482/743. In some embodiments, the isolated peptide can comprise theamino acid sequence of SEQ ID NO: 10 identified herein, and the methodcan comprise monitoring precursor-product ion pair transitions having anm/z value of about 491/721, 491/834, or 491/535. In some embodiments,the isolated peptide can comprise the amino acid sequence of SEQ ID NO:11 identified herein, and the method can comprise monitoringprecursor-product ion pair transitions having an m/z value of about528/501, 528/614, or 528/727. In some embodiments, the isolated peptidecan comprise the amino acid sequence of SEQ ID NO: 12 identified herein,and the method can comprise monitoring precursor-product ion pairtransitions having an m/z value of about 571/783, 571/971, 571/1028,381/587, 381/474, or 381/375. In some embodiments, the isolated peptidecan comprise the amino acid sequence of SEQ ID NO: 13 identified herein,and the method can comprise monitoring precursor-product ion pairtransitions having an m/z value of about 695/695, 695/837, or 695/950.In some embodiments, the isolated peptide can comprise the isolatedpeptide comprises the amino acid sequence of SEQ ID NO: 14 identifiedherein, and the method can comprise monitoring precursor-product ionpair transitions having an m/z value of about 536/795, 536/584, or536/698.

According to various embodiment the method can comprise determining anamount of CYP 3A5 in the sample by detecting an isolated peptidespecific to cytochrome P450 (CYP) isoform CYP 3A5, comprising the aminoacid sequence of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17identified herein. The method can use, for example, a triple quadrupoleinstrument and Multiple Reaction Monitoring (MRM). In some embodiments,the isolated peptide can comprise an amino acid sequence of SEQ ID NO:15 identified herein, and the method can comprise monitoringprecursor-product ion pair transitions having an m/z value of about468/581, 468/679, or 468/736. In some embodiments, the isolated peptidecan comprise on amino acid sequence of SEQ ID NO: 16 identified herein,and the method can comprise monitoring precursor-product ion pairtransitions having an m/z value of about 470/494, 470/608, or 470/722.In some embodiments, the isolated peptide can comprise an amino acidsequence of SEQ ID NO: 17 identified herein, and the method can comprisemonitoring precursor-product ion pair transitions having an m/z value ofabout 589/747, 589/696, or 589/647.

According to various embodiments of the present teachings, a kit isprovided that can comprise one or more of the isolated peptides specificto one or more of cytochrome P450 (CYP) isoform CYP 2B6, CYP 3A4, CYP1A2, and CYP 3A5. For example, the kit can comprise one or more isolatedproteins specific to CYP 2B6, comprising the amino acid sequence of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The kit cancomprise one or more isolated proteins specific to CYP isoform CYP 3A4,comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQID NO: 7. The kit can comprise one or more isolated proteins specific toCYP isoform CYP 1A2, comprising the amino acid sequence of SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO: 14. The kit can comprise one or more isolated proteinsspecific to CYP isoform CYP 3A5, comprising the amino acid sequence ofSEQ ID NO: 15, SEQ ID NO 16, or SEQ ID NO: 17.

In some embodiments, the kit can comprise at least one isolated peptidespecific to each of CYP isoforms CYP 2B6, CYP 3A4, CYP 1A2, and CYP 3A5.For example, the kit can comprise each of the isolated peptides of SEQID NOS: 1-17 identified herein, and further can comprise instructionsfor measuring Q1 and Q3 transition values for each of theisoform-specific peptides. The kit can comprise enzyme digestioncomponents including buffers and enzymes, other buffers, and optionallyother reagents and/or components. In some embodiments, the kit cancomprise, for example, a homogeneous assay such that the user need onlyadd a sample. In some embodiments, the kit can comprise calibration ornormalization reagents or standards. Information pertaining toinstrument settings that can or should be used to perform an assay canalso be included in the kit. Information pertaining to samplepreparation, operating conditions, volumetric amounts, temperaturesettings, and the like, can be included with the kit.

According to various embodiments, different transitions can be used tomeasure and benchmark assay results, depending on various factors.Accordingly, the kit can comprise different transition values and/orsuggested settings, useful to make comparative measurements between asample and one or more control reagents. The kit can includeinstructions to measure specific pairs of transition values, forexample, the Q1/Q3 transition pair, or the values of one or moredifferent transition pairs.

The kit can be packaged in a hermetically sealed container containingone or more regent vessels and appropriate instructions. An electronicmedium can be included in the kit, having stored thereon electronicinformation pertaining to one or more assays, measurement values,transition pairs, operating instructions, software for carrying outoperations, a combination thereof, or the like.

EXAMPLES

The present teachings can be even more fully understood with referenceto the examples and resulting data that follow. In the examples belowand the results shown in the attached drawing figures, a CYP inductionstudy was undertaken as follows.

Hepatocyte Treatment

Primary cultures of humus hepatocytes were treated for 72 hours with theprototypical liver enzyme inducers 3-methylcholanthrene (3-MC, 2 μM),phenobarbital (PB, 1 mM), or rifampicin (RIF, 10 μM) to enhance theexpression and activity of CYP1A2, CYP2B6, and CYP3A4, respectively. TheCYP quantitation method of the present teachings was used to determinethe amount of CYP in the sample after treatment with the liver enzymeinducers. In addition, CYP activity was measured by metabolite formationfrom selective substrates (phenacetin, bupropion and testosterone,respectively) and mRNA was measured by qRT-PCR (Taqman®, AppliedBiosystems).

Microsome Preparation

Microsomal subcellular fractions were prepared by lysing treatedhepatocytes in homogenization buffer (50 mM TRIS-HCl, pH 7.0, 150 mMKCl, 2 mM EDTA) followed by centrifugation at 9,000×g for 20 minutes at4° C. The supernatant (S9 fraction) was then spun at 100,000×g for 60minutes at 4° C. The resulting microsomal pellet was resuspended in 0.25M sucrose and stored at −80° C. until analysis.

Tryptic Digestion

To 100 ul of each microsomal preparation, 5 ul 2% SDS was added,followed by 10 ul of 50 mM TCEP and incubated at 60° C. for 1 hr, 5 ulof 0.1M MMTS was added and incubated at room temperature for 10 minutes.100 ul of 100 mM TRIS (pH=8.50) was then added followed by 50 ug oftrypsin and the resultant solution was digested overnight at 37° C. On(1) ng of each isotopically enriched synthetic peptide was added to thedigest and analyzed by LC-MS.

Chromatography

Chromatography was performed using an Agilent 1100 system (Agilent ofSanta Clara, Calif.) coupled to a C18 Jupiter Proteo 50×2.0 mm column(Phenomenex of Torrance, Calif.). The gradient was 5-40% B over 15minutes with A consisting of 2% ACN, 0.1% formic and B consisting of 90%ACN, 10% H2O, 0.1% formic acid. Flow rate was 700 μL/min.

Mass Spectrometry

Samples were analyzed on a Applied Biosystems MDS SCIEX 4000 QTRAP™LC/MS/MS system, using a Turbo V™ source and Analyst 1.5. Forquantitation, scheduled MRM (sMRM) was used to maximize dwelt time oneach transition.

Data Processing

Quantitative data was processed using MultiQuart™ 1.2 software availablefrom Applied Biosystems, LLC of Foster City, Calif.

Results

FIG. 6 is a graph comparing the changes in expression of CYP 2B6observed using the RNA assay, the CYP activity assay (designated “enzymeactivity assay” in the figures), and a CYP quantitation method of thepresent teachings (designated “protein assay” in the figures). The 3-MCis a vehicle control and induces basal levels of CYP 2B6. The PB is aprototypical inducer for CYP 2B6, by CAR nuclear inceptor activation.The RIF is also a known inducer of CYP 2B6. As is shown, proteinexpression changes observed in the CYP activity assay and RNA assaygenerally mirror the expression changes observed using a CYPquantitation method of the present teachings.

FIG. 7 is a graph comparing the changes in expression of CYP 1A2observed using the RNA assay, the CYP activity assay (designated “enzymeactivity assay” in the figures), and the CYP quantitation method of thepresent teachings (designated “protein assay” in the figures). The 3-MCis a prototypical inducer of CYP 1A2 by AhR nuclear receptor activation.The PB and RIP are vehicle controls inducing basal levels of 1A2, ifany. As is shown, protein expression changes observed in the CYPactivity assay and RNA assay generally mirror the expression changesobserved using a CYP quantitation method of the present teachings,except that the RNA way for the sample treated with 3MC exhibits veryhigh levels of RNA. The RNA assay cannot accurately quantify proteinsbecause not all mRNA is converted to protein.

FIG. 8 is a graph comparing the changes in expression of CYP 3A4observed using the RNA assay, the CYP activity assay (designated “enzymeactivity assay” in the figures), and a CYP quantitation method of thepresent teachings (designated “protein assay” in the figures). The 3-NICminimally induces CYP 3A4. The PB significantly inducts 3A4. The RIF isa prototypical inducer of CYP 3A4 by PXR nuclear receptor activation. Asis shown, protein expression changes observed in the CYP activity anyand RNA assay generally mirror the expression changes observed using theCYP quantitation method of the present teachings.

Table 1 below shows sequences of the peptides determined, according tothe present teachings, to be specific to cytochrome P450 (CYP) isoformCYP 2B6, along with their optimal MRM Q1, Q3 transitions. According tovarious embodiments, these observed peptides and transitions can be usedto enable a reliable CYP quantitation of the isoform CYP 2B6.

TABLE 1 2B6 Human O1 O3 IAMVDPFFR 548.3 911.4 (SEQ ID NO: 1) 548.3 681.3548.3 566.3 IPPTYQIR 494.3 777.4 (SEQ ID NO: 2) 494.3 437.7 494.3 874.5FSVTTMR 421.2 508.4 (SEQ ID NO: 3) 421.2 607.3 421.2 694.4 ETLDPSAPK479.2 499.3 (SEQ ID NO: 4) 479.2 614.3 479.2 727.4

Table 2 below shows sequences of the peptides determined, according tothe present teachings, to be specific to cytochrome P450 (CYP) isoformCYP 3A4, along with their optimal MRM Q1, Q3 transitions. According tovarious embodiments, these observed peptides and transitions can be usedto enable a reliable CYP quantitation of the isoform CYP 3A4.

TABLE 2 3A4 Human O1 O3 EVTNFLR 439.7 549.3 (SEQ ID NO: 5) 439.7 650.5439.7 532.3 LSLGGLLQPEKPVVLK 704.4 794.5 (SEQ ID NO: 6) 704.4 929.8LSLGGLLQPEKPVVLK +3 564.3 789.5 (SEQ ID NO: 6) 564.3 745.9 564.3 689.4VWGFYDGQQPVLAITDPDMIK 798.4 819.4 (SEQ ID NO: 7) 798.4 932.5 798.41003.5

Table 3 below snows sequences of the peptides determined, according tothe present teachings, to be specific to cytochrome P450 (CYP) isoformCYP 1A2, along with their optimal MRM Q1, Q3 transitions. According tovarious embodiments, these observed peptides and transitions can be usedto enable a reliable CYP quantitation of the isoform CYP 1A2.

TABLE 3 1A2 Human O1 O3 DITGALFK 432.7 636.4 (SEQ ID NO: 8) 432.7 535.3432.7 478.3 YGDVLQIR 482.3 800.5 (SEQ ID NO: 9) 482.3 628.4 482.3 743.4FLWFLQK 491.3 721.4 (SEQ ID NO: 10) 491.3 834.4 491.3 535.3 ASGNLIPQEK528.7 501.2 (SEQ ID NO: 11) 528.7 614.4 528.7 727.4 IGSTPVLVLSR +2 571.4783.5 (SEQ ID NO: 12) 571.4 971.6 571.4 1028.6 IGSTPVLVLSR +3 381.1587.4 (SEQ ID NO: 12) 381.1 474.3 381.1 375.2 SPPEPWGWPLLGHVLYGK 695.4695.9 (SEQ ID NO: 13) 695.4 837.5 695.4 950.5 YLPNPALQR 536.3 795.4 (SEQID NO: 14) 536.3 584.3 536.3 698.4

Table 4 below shows sequences of the peptides determined, according tothe present teachings, to be specific to cytochrome P450 (CYP) isoformCYP 3A5, along with their optimal MRM Q1, Q3 transitions. According tovarious embodiments, these observed peptides and transitions can be usedto enable a reliable CYP quantitation of the isoform CYP 3A5.

TABLE 4 3A5 O1 O3 SLGPVGFMK 468.5 581.5 (SEQ ID NO: 15) 468.5 678.1468.5 735.5 DTINFLSK 469.5 494.3 (SEQ ID NO: 16) 469.5 608.4 469.5 721.5GSMVVIPTYALHHDPK 589.2 746.5 (SEQ ID NO: 17) 589.2 696 589.2 646.5

Table 5 below shows sequences of the peptides along with their optimalMRM Q1, Q3 transitions for the house-keeping microsomal proteinMicrosomal GST. According to various embodiments, the concentration ofthis observed peptide is unaffected by drug incubation and thus thepeptide can be useful as a normalization protein to enable reliable CYPquantitation.

TABLE 5 Microsomal GST O1 O3 DVNVENVNQQR 657.8 758.3 (SEQ ID NO: 18)657.8 887.5 657.8 545.3 MYLLALK 426.3 720.5 (SEQ ID NO: 19) 426.3 557.4426.3 444.3 NALLPEGIPSLLK 682.9 1066.7 (SEQ ID NO: 20) 682.9 727.5 682.9953.6

Table 6 below shows sequences of the peptides along with their optimalMRM Q1, Q3 transitions for the house-keeping microsomal proteinMicrosomal Triglyceride. According to various embodiments, theconcentration of this observed peptide is unaffected by drug incubationand thus the peptide can be useful as a normalization protein to enablereliable CYP quantitation.

TABLE 6 Microsomal Tryglyceride O1 O3 MMLMSTATAFYR 711.8 829.4 (SEQ IDNO: 21) 711.8 916.2 711.8 1047.5

Table 7 below shows sequences of the peptides along with their optimalMRM Q1, Q3 transitions for the house-keeping microsomal proteinCorticosteroid 11 beta. It should be understood that the Q1 and Q3masses for the peptide of SEQ ID NO: 22 in Table 7 refers to the MMTSalkylated peptide and that changing to a different alkylating reagentwill change the Q1, Q3 masses. In addition, alkylating reagents otherthan MMTS can be used. According to various embodiments, theconcentration of this observed peptide is unaffected by drug incubationand thus the peptide can be useful as a normalization protein to enablereliable CYP quantitation.

TABLE 7 Corticosteroid 11 beta O1 O3 EECALEIIK 547.3 615.4 (SEQ ID NO:22) 547.3 686.4 547.3 835.4 FALDGFFSSIR 630.3 928.4 (SEQ ID NO: 23)630.3 1041.5 630.3 813.4

The observed isoform-specific tryptic peptides that are detected usingLC-MSMS analysis of microsomes, along with their optimal Q1, Q3transitions, enable a method for CYP quantitation of the isoforms 1A2,2B6, 3A4, and 3A5 without the need for any chemical labeling approaches.

Other embodiments of the present teachings will be apparent to theskilled in the art from consideration of the present specification andpractice of the present teachings disclosed herein. It is intended thatthe present specification and examples be considered as exemplary only.

1. A method for determining an amount of at least one isoform ofcytochrome P450 (CYP) in a sample, said method comprising subjectingsaid sample to an analysis with a liquid chromatograph tandem massspectrometer system, said system comprising a triple quadrupoleinstrument and Multiple Reaction Monitoring (MRM), said analysiscomprising detecting at least one isolated peptide of CYP 3A4, CYP 1A2or CYP 3A5.
 2. The method of claim 1 wherein the inform is CYP 3A4 andsaid isolated peptide has the amino acid sequence of SEQ ID NO: 5, SEQID NO: 6, or SEQ ID NO: 7
 3. The method of claim 2, wherein the aminoacid sequence is SEQ ID NO:5 and said analysis is performed bymonitoring a precursor-product ion pair transition having an m/z valueof about 440/549, 440/650, or 440/532 in said system and processing datafrom said monitoring to determine said amount.
 4. The method of claim 2,wherein the amino acid sequence is SEQ ID NO:6 and said analysis isperformed by monitoring a precursor-product ion pair transition havingan m/z value of about 704/794, 704/929, 564/689, 564/745, or 564/790 insaid system and processing data from said monitoring to determine saidamount.
 5. The method of claim 2, wherein the amino acid sequence is SEQID NO:7 and said analysis is performed by monitoring a precursor-production pair transition having an m/z value of about 798/819, 798/932, or798/1004 in said system and processing data from said monitoring todetermine said amount.
 6. The method of claim 1 wherein the isoform isCYP 1A2 and said isolated peptide has the amino acid sequence of SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13 or SEQ ID NO:
 14. 7. The method of claim 6, wherein the aminoacid sequence is SEQ ID NO:8 and said analysis is performed bymonitoring a precursor-product ion pair transition having an m/z valueof about 432/636, 432/535, or 432/478 in said system and processing datafrom said monitoring to determine said amount.
 8. The method of claim 6,wherein the amino acid sequence is SEQ ID NO:9 and said analysis isperformed by monitoring a precursor-product ion pair transition havingan m/z value of about 482/800, 482/628, or 482/743 in said system andprocessing data from said monitoring to determine said amount.
 9. Themethod of claim 6, wherein the amino acid sequence is SEQ ID NO:10 andsaid analysis is performed by monitoring a precursor-product ion pairtransition having an m/z value of about 491/721, 491/834, or 491/535 insaid system and processing data from said monitoring to determine saidamount.
 10. The method of claim 6, wherein the amino acid sequence isSEQ ID NO:11 and said analysis is performed by monitoring aprecursor-product ion pair transition having an m/z value of about528/501, 528/614, or 528/727 in said system and processing data fromsaid monitoring to determine said amount.
 11. The method of claim 6,wherein the amino acid sequence is SEQ ID NO:12 and said analysis isperformed by monitoring a precursor-product ion pair transition havingan m/z value of about 571/783, 571/971, 571/1028, 381/587, 381/474, or381/375 in said system and processing data from said monitoring todetermine said amount.
 12. The method of claim 6, wherein the amino acidsequence is SEQ ID NO:13 and said analysis is performed by monitoring aprecursor-product ion pair transition having an m/z value of about695/695, 695/837, or 695/950 in said system and processing data fromsaid monitoring to determine said amount.
 13. The method of claim 6,wherein the amino acid sequence is SEQ ID NO:14 and said analysis isperformed by monitoring a precursor-product ion pair transition havingan m/z value of about 536/795, 536/584, or 536/698 in said system andprocessing data from said monitoring to determine said amount.
 14. Themethod of claim 1 wherein the isoform is CYP 3A5 and said isolatedpeptide has the amino acid sequence of SEQ ID NO: 15, SEQ ID NO 16, orSEQ ID NO: 17
 15. The method of claim 14, wherein the amino acidsequence is SEQ ID NO:15 and said analysis is performed by monitoring aprecursor-product ion pair transition having an m/z value of about468/581, 468/679, or 468/736 in said system and processing data fromsaid monitoring to determine said amount.
 16. The method of claim 14,wherein the amino acid sequence is SEQ ID NO:16 and said analysis isperformed by monitoring a precursor-product ion pair transition havingan m/z value of about 470/494, 470/608, or 470/722 in said system andprocessing data from said monitoring to determine said amount.
 17. Themethod of claim 14, wherein the amino acid sequence is SEQ ID NO:17 andsaid analysis is performed by monitoring a precursor-product ion pairtransition having an m/z value of about 589/747, 589/696, or 589/647 insaid system and processing data from said monitoring to determine saidamount.
 18. A method for screening a drug for cytochrome P450 (CYP)induction, comprising: incubating the drug with a microsome-containingbiological sample; determining a quantitated value of at least one of aCYP 3A4, CYP 1A2 or CYP 3A5 isoform in said drug incubated with themicrosome-containing biological sample, said determining comprising,subjecting said drug incubated with the microsome-containing biologicalsample to an analysis with a liquid chromatography tandem massspectrometer system, said system comprising a triple quadrupoleinstrument and multiple reaction monitoring, said analysis comprisingdetecting an isolated peptide of said isoform; comparing the quantitatedvalue to a threshold value; determining that the drug has an acceptableCYP induction potential when the quantitated value does not exceed thethreshold value.